Category: Preclinical Development
Purpose: Equilibrium dialysis (ED) has been widely used for the measurement of the fraction of unbound drug (fu) in different matrices such as plasma, liver microsomes, and tissue homogenate. Highly bound and slow-to-reach equilibrium compounds often require prolong incubation time to obtain accurate results, and thus concerns regarding matrix integrity arise. To address these concerns, we have investigated matrix integrity in a wide range of compounds by comparing fu of these compounds under two different matrices’ storing conditions for 18 hours followed by 6 hours of equilibrium dialysis.
Methods: Three matrices were investigated in this study including plasma, liver microsomes, and brain homogenates. We have included a variety of compounds in this study containing acidic, basic and neutral compounds. Each of the matrices was separated into two portions and stored in two different conditions, one in the shaking incubator at 37ºC with 5% CO2,and second 4ºC in the fridge, for 18 hours prior to incubation for equilibrium dialysis. Subsequently, individual drugs were spiked into the two sets matrices separately to achieve a final concentration of 5 µM, and then 300 µL of drug-plasma mixtures were transferred to the donor wells of the RED plate which was pre-loaded with 500 µL phosphate buffer saline on the receiver wells. The RED plate was sealed and placed in a shaking incubator for 6 hr at 37ºC with 5% CO2. At the end of incubation, aliquots of samples were taken out of the RED device and matrix equalized with an equal volume of matrix or buffer, and samples were then immediately quenched with ice cold acetonitrile (sample : acetonitrile 1:3) containing internal standards. After shaking, all samples were then subjected to centrifugation to remove protein. Subsequently, supernatants were collected and then diluted with an equal volume of water prior to LC-MS/MS analysis. The two sets of fu values were then calculated and compared.
Results: Our results clearly showed that there is high correlation between the two sets of fu values obtained from the two conditions in all three matrices. The R2 values calculated from the two sets of fu measured are 0.983, 0.978, and 0.951 in plasma, liver microsomes, and brain homogenates respectively. The results indicate prolong incubation at 37ºC does not change the binding property, suggesting the matrix integrity is preserved.
Conclusion: The results indicate the integrity of the matrices are well maintained, and support the use of longer incubation time for the compounds that take longer time to reach equilibrium.